Oxidation/isomerization of 5-cholesten-3 beta-ol and 5-cholesten-3-one to 4-cholesten-3-one in pure sterol and mixed phospholipid-containing monolayers by cholesterol oxidase.

In this study we have examined the cholesterol oxidase (Streptomyces cinnamomeus) catalyzed conversion of either 5-cholesten-3 beta-ol or 5-cholesten-3-one into 4-cholesten-3-one in pure sterol or mixed phospholipid-containing monolayers at the air/buffer interface. The mean molecular area requirement of 5-cholesten-3-one in a pure monolayer was slightly smaller than the comparable area required by 5-cholesten-3 beta-ol (although the collapse pressure was markedly lower for 5-cholesten-3-one), and both sterols were about equally capable of condensing the lateral packing density of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at a lateral surface pressure of 20 mN/m. Both sterols were converted by cholesterol oxidase to 4-cholesten-3-one, the reaction being faster with 5-cholesten-3-one as compared to 5-cholesten-3-beta-ol. When the temperature-dependency of the cholesterol oxidase catalyzed conversion of the sterols to 4-cholesten-3-one was examined, the Arrhenius activation energy was calculated to +30 kJ/mol and +27 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively, when the sterols were presented to the enzyme as pure sterol monolayers at a lateral surface pressure of 20 mN/m. With a mixed monolayer containing 40 mol% sterol and 60 mol% EPC, the corresponding activation energies were +107 kJ/mol and +96 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively. With the monolayer system used, it appeared that the over all rate-limiting step in the enzyme-catalyzed conversion of 5-en-sterols to 4-en-3-one was the desorption of the sterol molecules from the monolayer into the active site of the enzyme at the interface. This appeared to be true both with pure sterol monolayers as well as with mixed monolayers containing phosphatidylcholine.